
==== Front
STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00445-3
10.1016/j.xpro.2024.103280
103280
Protocol
Protocol for generating human cortical organoids enriched in outer radial glia by guided differentiation
Luongo Raffaele raffaele.luongo@ior.usi.ch
125∗
Walsh Ryan M. 3
Verrillo Antonietta 12
Studer Lorenz 34
Baggiolini Arianna arianna.baggiolini@ior.usi.ch
126∗∗
1 Institute of Oncology Research (IOR), Bellinzona Institutes of Science (BIOS+), 6500 Bellinzona, Switzerland
2 Faculty of Biomedical Sciences, Università della Svizzera Italiana, 6900 Lugano, Switzerland
3 Center for Stem Cell Biology and Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
4 Weill Cornell Medicine Graduate School of Medical Sciences, Department of Neuroscience, New York, NY 1300, USA
∗ Corresponding author raffaele.luongo@ior.usi.ch
∗∗ Corresponding author arianna.baggiolini@ior.usi.ch
5 Technical contact

6 Lead contact

29 8 2024
20 9 2024
29 8 2024
5 3 103280© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

The generation of human pluripotent stem cell (hPSC)-derived brain organoids is continuously refined, enhancing their reproducibility and complexity. Here, we present a guided differentiation protocol for generating cortical forebrain organoids and cortico-pericyte (CP)assembloids composed of a robust outer radial glia (oRG) population and an expanded outer subventricular zone (oSVZ). We describe the steps to generate hPSC-derived cortical organoids (COs), cortical pericytes, and CP assembloids. Moreover, we outline the procedures to characterize the organoids by immunostaining and to perform single-cell dissociation.

For complete details on the use and execution of this protocol, please refer to Walsh et al.1

Graphical abstract

Highlights

• Guided differentiation protocol with LIF treatment for cortical organoids (COs)

• Steps for generating LIF-secreting cortical pericytes and CP assembloids

• Guide to cryosectioning and immunofluorescence of COs and CP assembloids

• Instructions for single-cell dissociation of organoids and assembloids

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The generation of human pluripotent stem cell (hPSC)-derived brain organoids is continuously refined, enhancing their reproducibility and complexity. Here, we present a guided differentiation protocol for generating cortical forebrain organoids and cortico-pericyte (CP) assembloids composed of a robust outer radial glia (oRG) population and an expanded outer subventricular zone (oSVZ). We describe the steps to generate hPSC-derived cortical organoids (COs), cortical pericytes, and CP assembloids. Moreover, we outline the procedures to characterize the organoids by immunostaining and to perform single-cell dissociation.

Subject areas

Cell Biology
Cell culture
Cell Differentiation
Cell isolation
Developmental biology
Flow Cytometry
Neuroscience
Organoids
Stem Cells
==== Body
pmcBefore you begin

Human pluripotent stem cell (hPSC)-derived brain organoids are 3D structures that faithfully recapitulate some specific aspects of tissue architecture and cellular composition of the human brain. Brain organoids can be generated following either unguided differentiation protocols or guided differentiation strategies based on the addition of small molecules and growth factors to generate region-specific areas of the brain.2

The developing human brain arises from neural progenitor populations that are enriched in the ventricular zone (VZ) and subventricular zone (SVZ).3 The SVZ can be further divided into inner SVZ (iSVZ) and outer SVZ (oSVZ). The outer radial glia (oRG) population is enriched in the expanded oSVZ of gyrencephalic species (e.g., human), while it’s mainly absent in lissencephalic species (e.g., mouse), suggesting a potential role in neocortex expansion and folding.4

In current guided CO differentiation protocols, oRG cells are rare, with only a few cells co-expressing oRG markers, such as Glial Fibrillary Acid Protein (GFAP) and HOP Homeobox (HOPX). Conversely, unguided brain organoid differentiation protocol can eventually lead to the spontaneous generation of oRG.1,5 To faithfully recapitulate human-specific aspects of brain development, it is crucial to recapitulate the presence of a robust oRG population also in cortical organoids derived from guided differentiation protocols.

The protocol described in this manuscript derives from a previously published protocol to generate cortical forebrain organoids6 and is adapted to promote the emergence of oRG and the expansion of the oSVZ.

This protocol allows the generation of a robust oRG population by either treatment with Leukemia Inhibitory Factor (LIF) or the incorporation of cortical LIF-secreting pericytes into COs to generate the CP assembloids.

We will show, in detail, the steps to generate COs, cortical pericytes and CP assembloids, how to characterize them by immunofluorescence and how to dissociate them into viable single cells for further analysis as we show in Walsh et al.1

To generate oRG-enriched COs, cortical pericytes and CP assembloids it is essential to procure all necessary materials and equipment listed in the key resource table (key resources table).

The hPSCs used in this protocol are maintained in Essential 8 (E8) medium (Thermo Fisher Scientific, #A1517001) and, in case you are using a different medium for your hPSCs culture, we recommend switching to E8 for the successful reproduction of the described protocol. Moreover, hPSCs are cultured in the absence of any antibiotics, so it is essential to use standard sterilized equipment and maintain the environment sterile by working in a Class II Biohazard hood. The cells and the organoids must be cultured in a tissue culture incubator with a 37°C temperature and a 5% CO2 atmosphere.

All the procedures have been validated by using human embryonic stem cell (hESC) lines, such as WA09 and WA01 (from WiCell), and human induced pluripotent stem cell (hiPSC) lines, e.g., the MSK-SRF0015 and CS5DZLiCTR-n5 (from https://data.answerals.org/home ) lines, with comparable results.

Before starting any experiment, it is essential to secure the necessary approvals to work with hESCs or patient-derived hiPSCs lines. This includes obtaining the appropriate approvals from the Institutional Review Board (IRB), Ethics Commissions, and ensuring that Material Transfer Agreements (MTAs) are in place for any material obtained from external sources. These precautions are important for ensuring that the research is conducted ethically and in compliance with regulatory guidelines, as done in the reference study.1Note: If you choose to use reagents from different suppliers, please be mindful to verify that the differentiation efficiency is not altered.

Prepare vitronectin-coated plates

Timing: 45 min

1. Thaw one aliquot of Vitronectin Recombinant Human Protein, Truncated (rh-VTN) (Thermo Fisher Scientific, #A14700) at room temperature (RT) until is completely melted.a. Upon arrival, rh-VTN must be stored at −80°C.

Note: If you are using the 10 mL bottle (Thermo Fisher Scientific, #A31804), thaw it, make aliquots of the required volume, and store them at −80°C for maximum 6 months or until the recommended expiration date.

2. Dilute the rh-VTN 1:100 in sterile DPBS without calcium and magnesium (Thermo Fisher Scientific, #14190094) to prepare the coating working solution.

Note: From now on, unless it’s differently specified, DPBS is without calcium and magnesium.

3. Coat plates by adding an amount of diluted rh-VTN sufficient to cover the entire surface of the plate.a. For 100-mm dishes, use at least 6 mL.

b. For 60-mm dishes, use 2 mL.

c. For 6-well plates, use 1 mL for each well.

Note: If you are pre-preparing the plates with the intention of storing them (step 5), consider increasing the volume of coating solution to avoid the plates drying out due to liquid evaporation.

4. Swirl the plate to spread the coating solution over its entire surface area.

5. Leave the plate at RT under the biological hood for a minimum of 30 min before using it.

Note: If not used immediately, seal the cultureware with parafilm and store it at 4°C for up to 2 weeks.

Prepare the EDTA solution

Timing: 1 min

6. Prepare 0.5 mM DPBS/EDTA solution by diluting 1:1000 the UltraPure™ 0.5 M EDTA pH 8.0 (Thermo Fisher Scientific, #15575020) into DPBS.a. For 50 mL of solution, dilute 50 μL of EDTA in 50 mL of DPBS.

7. Sterilize the solution using a 0.22 μM filter.

Culture of the hPSCs

Timing: 4–5 days for cells to reach the required confluency;10 min for passaging

8. Grow hESCs and hiPSCs in Essential 8 (E8) medium on rh-VTN coated plates and incubate at 37°C with 5% CO2.a. Perform daily medium change, completely replacing the medium with fresh E8.

Note: If you culture hPSCs in media other than E8, we suggest adapting cells to E8 medium before starting the differentiation for the complete success of the described differentiation protocols.

CRITICAL: The differentiation efficiency strongly depends on the hPSCs quality. Avoid letting hPSCs become overconfluent and check for the presence of contaminant differentiated cells.

9. Passaging hPSCs using EDTA solution.Note: The subsequent steps describe how to culture cells in a 60-mm dish. Adjust volume accordingly to scale up or down depending on the size of the dish chosen.

a. Before you begin, prepare rh-VTN coated plates or place them at RT if previously prepared and stored at 4°C.

b. Remove all the medium from the hPSCs plate and rinse cells once with 2.5 mL of 1× DPBS to wash away the Mg2+ and Ca2+ from the E8 medium.

c. Add 1 mL of EDTA solution and swirl the plate to cover the entire surface area of the plate.Note: ReLeSR (StemCell Technologies #100-0483) is a valid alternative to EDTA solution for passaging hPSCs. Use it according to manufacturer instructions.

d. Incubate the plate for 3–5 min at 37°C.CRITICAL: Check cells under the microscope regularly to avoid their prolonged exposure to EDTA solution. Stop incubation when the border of the hPSC colonies becomes translucent. Longer periods in EDTA can lead to colonies that are too small or even single cells.

e. While cells are incubating with the EDTA solution, remove the rh-VTN coating solution from the plates, label the plates, and add 3 mL of E8 medium.

f. When colonies start to appear translucent, carefully aspirate the EDTA solution, avoiding aspirating also the cells, and add with a strong flow 1 mL of E8 medium to facilitate dispersing the colonies in the dish.

g. Tap the side of the dish with the finger to dislodge the colonies from the plate.

h. Pipet the cell suspension up and down one or two times using a P1000 pipette to break the bigger colonies.CRITICAL: Do not pipet up and down with excessive vigor to avoid breaking the cell clumps into single cells. Never use 200 μL or smaller microliter pipette tips during these steps. The resulting sheer forces risk breaking the cell clusters into single cells.

i. Re-plate the hPSCs by transferring the required volume of the cell suspension to the new VTN-coated plate prefilled with E8 medium.Note: As a recommendation, the optimal splitting density is among 1:6 to 1:10 (based on the cell density and proliferation rate). This ratio should require the splitting of cells 2 times per week, likely every four days. Adjust the splitting ratio if your cells grow at different rates. Excessive dilution could impact the long-term quality of cells.

j. Position the plate in the incubator (37°C, 5% CO2) and move the plate forth and back to guarantee an equal distribution of cell clusters throughout the whole surface.

k. The next day, refresh the E8 medium.

10. Replace the medium and monitor the confluency of cells daily to avoid over-confluency and to check the presence of contaminant differentiated cells. Troubleshooting 1.

Prepare Matrigel-coated plates

Timing: 1 h

11. Thaw Matrigel (Corning, #354234) overnight (14–18 h) on ice at 4°C.

12. The next day, prepare 600 μL Matrigel aliquots.a. Matrigel aliquots can be stored at −80°C for up to 6 months.

Note: 600 μL aliquots are sufficient to prepare 18 mL of coating solution, which is enough for three 6-well plates. If using a different plate format, adjust the volumes accordingly.

CRITICAL: Always keep Matrigel on ice to prevent it from gelling.

13. Prepare the Matrigel-coating solution by diluting Matrigel aliquots 1:30 (for a final concentration of around 0.3–0.35 mg/mL) in cold DMEM/F-12 (Thermo Fisher Scientific, #31330038) and mix the solution well.a. 600 μL Matrigel aliquot must be diluted in 18 mL of DMEM/F12.

14. Coat the plate by adding 1 mL of Matrigel-coating solution into each well of a 6-well plate.a. 18 mL of Matrigel-coating solution allows the coating of three full 6-well plates.

15. Swirl the plate to spread the coating solution and completely cover the surface of the wells.

16. Incubate the Matrigel-coated plate at 37°C for a minimum of 30 min before using it.

Note: If not used immediately, Matrigel-coated plates can be sealed with parafilm and stored at 4°C for up to 2 weeks.

17. Before using the plate, wash it once with fresh DMEM/F12.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
SOX2 monoclonal antibody (Btjce), eBioscience (1:300)	Thermo Fisher Scientific	Cat#14-9811-82; RRID:AB_11219471	
EOMES monoclonal antibody (WD1928), eBioscience (1:300)	Thermo Fisher Scientific	Cat#14-4877-82; RRID:AB_2572882	
Recombinant anti-TBR1 antibody [EPR8138(2)] (1:400)	Abcam	Cat#ab183032; RRID:AB_2936859	
Anti pVIM (phospho-vimentin) (Ser55) (1:200)	MBL International	Cat#D076-3; RRID:AB_592963	
Anti-HOPX polyclonal antibody (1:200)	Sigma-Aldrich	Cat#HPA030180; RRID:AB_10603770	
GFAP antibody (1:400)	Novus Biologicals	Cat#NBP1-05198; RRID:AB_1556315	
CD271 (NGF receptor) monoclonal antibody (ME20.4), PE, eBioscience (1:50)	Thermo Fisher Scientific	Cat#12-9400-42; RRID:AB_2572710	
Donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 488 (1:500)	Thermo Fisher Scientific	Cat#A32790; RRID:AB_2762833	
Donkey anti-rat IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 555 (1:500)	Thermo Fisher Scientific	Cat#A48270; RRID:AB_2896336	
Donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (1:500)	Thermo Fisher Scientific	Cat#A31571; RRID:AB_162542	
Donkey anti-Rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 594 (1:500)	Thermo Fisher Scientific	Cat#A32754; RRID:AB_2762827	
Donkey anti-Chicken IgY (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (1:500)	Thermo Fisher Scientific	Cat#A78948; RRID:AB_2921070	
	
Chemicals, peptides, and recombinant proteins	
	
Essential 8 medium	Thermo Fisher Scientific	Cat#A1517001	
Vitronectin (VTN-N) recombinant human protein, truncated	Thermo Fisher Scientific	Cat#A14700	
UltraPure 0.5M EDTA, pH 8.0	Thermo Fisher Scientific	Cat#15575020	
ReLeSR	STEMCELL Technologies	Cat#100-0483	
Accutase solution	Sigma-Aldrich	Cat#A6964	
ROCK inhibitor Y-27632	Bio-Techne	Cat#1254	
Essential 6 medium	Thermo Fisher Scientific	Cat#A1516401	
LDN-193189	Reprocell (Stemgent)	Cat#04-0074	
SB 431542	Bio-Techne	Cat#1614	
XAV 939	Bio-Techne	Cat#3748	
Neurobasal medium	Thermo Fisher Scientific	Cat#21103049	
DMEM/F-12, HEPES	Thermo Fisher Scientific	Cat#31330038	
B-27 supplement (50×), minus vitamin A	Thermo Fisher Scientific	Cat#12587010	
N-2 supplement (100×)	Thermo Fisher Scientific	Cat#17502048	
GlutaMAX supplement	Thermo Fisher Scientific	Cat#35050038	
2-Mercaptoethanol	Thermo Fisher Scientific	Cat#21985023	
Normocin – antimicrobial reagent	InvivoGen	Cat#ant-nr-2	
Insulin solution human	Sigma-Aldrich	Cat#I9278	
Recombinant human LIF	PeproTech	Cat#300-05	
Matrigel basement membrane matrix, LDEV-free, 10 mL	Corning	Cat#354234	
Penicillin-Streptomycin	Sigma-Aldrich	Cat#P4333	
Recombinant human BMP-4 protein	Bio-Techne	Cat#314-BP	
CHIR 99021	Bio-Techne	Cat#4423	
DAPI	Sigma-Aldrich	Cat#D9542	
PM Pericyte medium	ScienCell Research Laboratories	Cat#1201	
Trypsin-EDTA (0.05%) in DPBS (1×)	Capricorn Scientific	Cat#TRY-1B	
OCT embedding matrix – 125 mL	CellPath	Cat#KMA-0100-00A	
Normal donkey serum	Abcam	Cat#ab7475	
Triton X-100	Sigma-Aldrich	Cat#T8787	
DPBS, calcium, magnesium	Thermo Fisher Scientific	Cat#14040091	
Tween 20	Sigma-Aldrich	Cat#P9416	
Fluoromount-G mounting medium	Thermo Fisher Scientific	Cat#00-4958-02	
Neutral buffered formalin	Epredia	Cat#5701	
DPBS, no calcium, no magnesium	Thermo Fisher Scientific	Cat#14190094	
Fetal bovine serum advanced (FBS advanced), collected in South America	Capricorn Scientific	Cat# FBS-11A	
Dimethyl sulfoxide, bioreagent, 250 mL	Thermo Fisher Scientific	Cat#J66650.AK	
Sucrose for molecular biology, ≥99.5% (GC)	Sigma-Aldrich	Cat# S0389	
	
Critical commercial assays	
	
Papain dissociation system	Worthington Biochemical	Cat#LK003150	
	
Experimental models: Cell lines	
	
WA09	WiCell	RRID:CVCL_9773	
WA01	WiCell	RRID:CVCL_9771	
MSK-SRF001	Generated in Li et al.7	N/A	
CS5DZLiCTR	https://data.answerals.org/home	N/A	
	
Software and algorithms	
	
BioRender	BioRender	www.biorender.com	
	
Other	
	
PrimeSurface 3D culture: ultra-low attachment plates: 96-well, V bottom, clear plates	S-Bio	Cat#MS-9096VZ	
Shaker, Light Duty, SHLD0415DG, EU	Ohaus	Cat#30391914	
Corning 100 mm TC-treated culture dish	Corning	Cat# 430167	
Falcon 60 mm TC-treated Easy-Grip Style cell culture dish	Falcon	Cat# 353004	
PELCO flat embedding mold, blue	Ted Pella	Cat#105	
Sterile disposable stainless steel scalpels	Swann-Morton	Cat#0501	

Materials and equipment

Day −1 medium

Reagent	Final concentration	Amount	Storage	
Essential 8	N/A	10 mL	4°C	
Y-27632 Rock Inhibitor [10 mM]	10 μM	10 μL	−20°C	
XAV 939 [10 mM]	5 μM	5 μL	−80°C	
Total	N/A	10 mL	4°C	
Note on storage: after the thawing, the small molecules can be stored at 4°C for up to one month.

CO 1 medium: Can be prepared and stored at 4°C for 1 week

Reagent	Final concentration	Amount	Storage	
Essential 6	N/A	50 mL	4°C	
LDN193189 [500 μM]	100 nM	10 μL	−80°C	
SB431542 [10 mM]	10 μM	50 μL	−80°C	
XAV 939 [10 mM]	5 μM	25 μL	−80°C	
Total	N/A	50 mL	4°C	
Note on storage: after the thawing, the small molecules can be stored at 4°C for up to one month.

CO 2 medium: Can be prepared and stored at 4°C for 1 week

Reagent	Final concentration	Amount	Storage	
Essential 6	N/A	30 mL	4°C	
LDN193189 [500 μM]	100 nM	6 μL	−80°C	
SB431542 [10 mM]	10 μM	30 μL	−80°C	
Total	N/A	30 mL	4°C	
Note on storage: after the thawing, the small molecules can be stored at 4°C for up to one month.

CO 3 medium: Can be prepared and stored at 4°C for 2–3 weeks

Reagent	Final concentration	Amount	Storage	
Neurobasal	N/A	235 mL	4°C	
DMEM/F12	N/A	235 mL	4°C	
Glutamax [100×]	1×	10 mL	RT	
B27 w/o vit. A [50×]	0.5×	10 mL	−20°C	
N2 [100×]	0.5×	5 mL	−20°C	
Insulin [9.5–11.5 mg/mL]	∼2.5 μg/mL	250 μL	4°C	
2-mercaptoethanol (2-BME) [55 mM]	0.055 mM	1 mL	4°C	
Normocin [1000×]	1×	1 mL	−20°C	
Total	N/A	500 mL	4°C	
LIF [100 μg/mL]	10 ng/mL	1 μL for 10 mL	−80°C	

CRITICAL: Filter with a 0.22 μm pore membrane. Do not freeze medium.

Note: It is suggested to prepare the medium in 500 mL-1 L volume. At every medium change, add fresh LIF in the needed amount. RT: Room Temperature (20°C–25°C).

NC 1 medium: Can be prepared and stored at 4°C for 1 week

Reagent	Final concentration	Amount	Storage	
Essential 6	N/A	10 mL	4°C	
BMP4 [10 μg/mL]	1 ng/mL	1 μL	−80°C	
SB431542 [10 mM]	10 μM	10 μL	−80°C	
CHIR 99021 [6 mM]	600 nM	1 μL	−80°C	
Total	N/A	10 mL	4°C	
Note on storage: after the thawing, the small molecules can be stored at 4°C for up to one month.

NC medium 2: Can be prepared and stored at 4°C for 1 week

Reagent	Final concentration	Amount	Storage	
Essential 6	N/A	10 mL	4°C	
SB431542 [10 mM]	10 μM	10 μL	−80°C	
CHIR 99021 [6 mM]	1.5 μM	2.5 μL	−80°C	
Total	N/A	10 mL	4°C	
Note on storage: after the thawing, the small molecules can be stored at 4°C for up to one month.

FACS medium: Can be prepared and stored at 4°C for 1 month

Reagent	Final concentration	Amount	Storage	
DMEM/F12	N/A	50 mL	4°C	
Fetal Bovine Serum (FBS)	2%	1 mL	−20°C	
0.5M EDTA	1 mM	50 μL	RT	
Normocin	500 μg/mL	50 μL	−20°C	
Total	N/A	50 mL	4°C	
RT: room temperature (20°C–25°C).

Freezing medium: Can be prepared and stored at 4°C for 1 month

Reagent	Final concentration	Amount	Storage	
Fetal Bovine Serum (FBS)	90%	45 mL	−20°C	
DMSO	10%	5 mL	RT	
Total	N/A	50 mL	4°C	
RT: room temperature (20°C–25°C).

Sucrose solution: Always prepare it fresh

Reagent	Final concentration	Amount	Storage	
Sucrose	30%	15 g	RT	
DPBS	N/A	50 mL	RT	
Total	N/A	50 mL	4°C	
RT: room temperature (20°C–25°C).

Blocking solution

Reagent	Final concentration	Amount	Storage	
DPBS with Ca2+ and Mg2+	N/A	19 mL	RT	
Triton-X-100	0.3%	60 μL	RT	
BSA	2%	0.4 g	4°C	
Donkey Serum	5%	1 mL	−20°C	
Total	N/A	20 mL	4°C	
RT: room temperature (20°C–25°C). Note on storage: to store the solution for more than 1 week at 4°C, add 0.02% sodium azide.

PBS-T 0.05% washing buffer

Reagent	Final concentration	Amount	Storage	
DPBS	N/A	500 mL	RT	
Tween-20	0.05%	250 μL	RT	
Total	N/A	500 mL	4°C	
RT: room temperature (20°C–25°C). Note on storage: the solution is stable at RT.

Secondary antibody diluent

Reagent	Final concentration	Amount	Storage	
DPBS	N/A	50 mL	RT	
Tween-20	0.05%	25 μL	RT	
BSA	2%	1 g	4°C	
Total	N/A	50 mL	4°C	
Note on storage: RT: Room temperature (20°C–25°C). To store the solution for more than 1 week at 4°C add 0.02% sodium azide.

DAPI solution

Reagent	Final concentration	Amount	Storage	
DPBS	N/A	50 mL	RT	
DAPI	1:1,000	50 μL	4°C	
Total	N/A	50 mL	4°C/dark	
Note on storage: DAPI aliquots must be stored at −20°C. Once thawed, an aliquot is stable at 4°C in the dark for up to six months.

Step-by-step method details

Plating hPSCs for cortical organoid (CO) generation

Timing: 30–45 min

This step describes the procedure to plate hPSCs into a low attachment V-bottom 96-well plate to generate COs.1. Grow hPSCs to 70–80% confluency.a. The well-defined borders of colonies and the absence of spontaneously differentiated cells are important for the success of the differentiation protocols (Figure 1A). Troubleshooting 1.

Note: The subsequent steps will be described for cells cultured in 60-mm dishes. Adjust volume accordingly to the size of the plate.

2. Detach hPSCs using Accutase.a. Aspirate the medium from cells.

b. Rinse cells with DPBS.

c. Add 1 mL of Accutase.

d. Incubate cells 5 min at 37°C.

e. After the incubation, dissociate colonies into single cells by pipetting with a P1000 pipette.

f. Collect the cell suspension into a 15 mL centrifuge tube and dilute the Accutase by adding DMEM/F12 to a final volume of 10 mL.

3. Centrifuge cells at 200 rcf for 3 min at RT (20°C–25°C).

4. Aspirate the supernatant and resuspend cells in 5 mL of Day −1 medium (E8 medium supplemented with 10 μM Y-27632 and 5 μM XAV939).

5. Count cells by using a cell counter.a. Take an aliquot of 20 μL of cell suspension from the previous step and mix it 1:1 with 20 μL of Trypan blue.i. You can also consider the use of Erythrosin B as alternative to Trypan blue.

b. Count cells using an automated cell counter. Troubleshooting 2.i. As an alternative, count cells manually using a Burker or Neubauer chamber.

c. Take the required volume of cell suspension to have 1 × 106 cells for each 96-well plate that will be used for generating organoids.i. Each well will contain an organoid, so a full 96-well plate allows to generate 96 organoids.

d. Dilute the 1 × 106 cell aliquot in Day −1 medium to a final volume of 10 mL.i. This will give you roughly 10 × 104 cells/100 μL.

6. Plate 1 × 104 cells in each well of a V-Bottom 96-well plate.a. Move the cell suspension to a sterile reservoir.

b. Using an 8-channel P200 pipette, plate 100 μL of cell suspension in each well of a V-Bottom 96-well plate.

7. Incubate the plate overnight (14–18 h) in a 37°C, 5% CO2 incubator, and avoid disturbance.

Figure 1 Generation of cortical organoids (COs) from hPSCs

(A) Representative bright field phase image showing the morphology of a hPSC colony. Scale bars: left panel, 400 μm; right panel, 300 μm.

(B) Schematical summary of the protocol used for the generation of COs.

(C) Bright field images showing the progressive changes in morphology of neural spheroids during the first 14 days of differentiation into COs. Scale bar: 400 μm.

(D) Bright field images of COs at day 32 and 45 of differentiation. Neuronal rosettes are evident at these stages. Scale bar: 500 μm.

Generating cortical organoids (COs)

Timing: 14 days

Here, three different media are used to provide an initial dual SMAD and Wnt inhibition to differentiate hPSCs into neuroectoderm and finally forebrain organoids. The protocol is schematized in Figure 1B.8. The day after the plating (Day 0), observe with a bright field microscope the successful formation of embryoid bodies (EBs) (Figure 1C).a. EBs have a spherical shape and smooth peripheral border. A small amount of cell debris around EBs will not affect the subsequent formation of organoids.

Note: If a large cloud of debris surrounds the organoid and covers more than 30% of the well surface, restart the differentiation. This might be due to a harsh hPSCs dissociation. Monitor cells during the Accutase step and, if needed, stop the incubation time earlier. Adopt gentle pipetting to collect the cells.

9. Change the medium to CO 1 medium.a. Using an 8-channel P200 pipette, remove 50 μL/well from the 96-well plate.

b. Add 100 μL of CO 1 medium to each well of the plate.

Note: The final volume in the well will be around 150 μL.

10. Change the medium daily with CO 1 medium until day 4.a. From day 1, the medium change is performed by removing 100 μL of medium and replacing it with 100 μL of fresh medium.

b. The CO 1 medium is used for a total of 5 days (from day 0 to day 4).

c. Monitor the sphere in the wells daily to ensure the lack of contamination and the correct growth of the organoids.

11. On day 5 of differentiation, change the medium and switch to the CO 2 medium.a. Remove 100 μL of medium and add 100 μL of CO medium 2.i. Organoids should appear bigger and with more defined borders (Figure 1C).

12. Change the medium daily with CO 2 medium until Day 7.a. Each medium change is performed by removing 100 μL of medium and replacing it with 100 μL of fresh medium.

b. The CO 2 medium is used for a total of 3 days (from day 5 to day 7).

13. On differentiation day 8, change medium, switching to CO 3 medium.a. Remove 100 μL of medium and add 100 μL of CO 3 medium.

b. It is recommended to add fresh LIF to the CO 3 medium at every medium change.CRITICAL: To generate organoids aimed at forming CP assembloids, LIF should not be added to the medium, since it’s going to be provided by the cortical pericytes.

c. Organoids are now bigger, with defined borders and a darker center (Figure 1C).

14. Change the medium every other day with CO 3 medium.a. Remove 100 μL of medium and add 100 μL of fresh CO 3 medium.

b. The CO 3 medium will be used until the end of the protocol.

15. At day 14, COs must be moved into 60- or 100-mm dishes and cultured at 37°C, 5% CO2 on an orbital shaker.a. At day 14 of differentiation, COs should appear more compact, with a darker and more defined border (Figure 1C).

Note: The shape of the organoids could be irregular and less spheroidal.

Growing cortical organoids (COs)

Timing: 45+ days

This phase of the protocol describes how to culture organoids in CO 3 medium on a shaking platform to allow them to grow and mature.16. Move the organoids from the 96-well plate into a 60- or 100-mm dish.Note: The size of the plate must be chosen considering the number of organoids you want to culture per plate and, more in general, the number of experimental conditions you plan to test.

a. Prepare a sufficient number of plates to culture all the organoids.i. 60-mm dishes can host a maximum of 8 COs.

ii. 100-mm dishes can host a maximum of 24 COs.Note: Increasing the number of COs in the dish can lead to their fusion Troubleshooting 3.

b. Add 6 mL or 25 mL of CO 3 medium to empty 60- or 100-mm dishes, respectively.

c. By using a sterile scalpel or scissors, cut the edge of a P1000 tip.Note: The tip should be cut to allow the organoid to be aspirated without damage.

d. Using a P1000 pipette with the cut tip, aspirate the CO from each V-bottom 96-well.i. Check the presence of the CO in the tip to ensure its aspiration from the well.

e. Release the organoid into pre-filled dishes of step b.

17. Culture the organoids at 37°C and 5% CO2 on an orbital shaker with an approximate rotation of 75 rpm.Note: 75 rpm is used for a shaker with a 15 mm orbit rotation. In case of different orbits, adjust the speed accordingly.

a. The 60-mm dishes require a faster rotation speed compared to the 100-mm dishes to avoid CO fusion; we recommend increasing the rotation to 85–90 rpm. Troubleshooting 3.

18. Perform medium change 3 times per week on a Monday, Wednesday, and Friday schedule.a. After tilting the plate on one side, use a 25 mL serological pipette to carefully aspirate 20 mL of medium (100-mm dishes) or 5 mL of medium (60-mm dishes). The plate tilting allows the medium to accumulate on one side, facilitating the medium aspiration and leaving the organoids untouched.

b. Add 20 mL or 5 mL of fresh CO 3 medium for 100-mm dishes and 60-mm dishes, respectively.

c. Position the plate back on the shaker into the incubator.

19. Monitor the COs growth during the maturation to ensure their correct patterning and the appearance of neuronal rosettes (Figure 1D). Troubleshooting 4.

Plating hPSCs for the neural crest (NC)-derived pericytes differentiation

Timing: 30 min

This procedure focuses on plating hPSCs to start a differentiation towards the cranial NC that, in turn, is differentiated into pericytes as schematized in Figure 2A.20. Before you begin, prepare Matrigel-coated 6-well plates or place them at RT if previously prepared and stored at 4°C.a. For this step of the protocol, more Matrigel-coated plates are required in 10 days. It is suggested having at least two 6-well plates ready for the next steps.

21. Grow hPSCs to 70–80% of confluency.a. Colonies with a healthy shape and the absence of spontaneously differentiated cells are important for the proper differentiation of hPSCs (Figure 1A). Troubleshooting 1.

Note: The subsequent steps will be described for cells cultured in 60-mm dishes. Adjust volume accordingly to scale up or down the size of the culture.

22. Detach hPSCs by Accutase.a. Aspirate the medium from cells.

b. Quickly rinse cells with DPBS.

c. Add 1 mL of Accutase.

d. Incubate cells 5 min at 37°C.

e. After incubation, dissociate colonies into single cells by pipetting with a P1000 pipette.

f. Collect the cell suspension into a 15 mL centrifuge tube and add 10 mL of DMEM/F12 to dilute the Accutase.

23. Centrifuge cells at 200 rcf for 3 min at RT (20°C–25°C).

24. Aspirate the supernatant and resuspend cells in 5 mL of Essential 8 medium supplemented with 10 μM of Y-27632.

25. Count cells by using a cell counter.a. To start a NC differentiation, cells should be plated as a high-density monolayer at 1.5 × 105 cells/cm2. The surface of a single well of a 6-well plate is 9.7 cm2, so 8.7 × 106 cells are needed for plating an entire 6-well plate.i. If you need more plates or fewer wells, adjust volumes accordingly.

b. Take an aliquot of 20 μL of cell suspension and mix it 1:1 with 20 μL of Trypan blue.i. You can also consider the use of Erythrosin B as alternative to Trypan blue.

c. Count cells by using an automated cell counter. Troubleshooting 2.i. As an alternative, count cells manually using a Burker or Neubauer chamber.

d. Take the needed volume of cell suspension to have 8.7 × 106 cells for each 6-well plate that will be used for generating NC.

26. Plate cells onto previously prepared Matrigel-coated 6-well plates.a. Dilute the cells into the needed volume of E8 medium supplemented with 10 μM Y-27632.i. For every full 6-well plate, use 1.5 mL of medium per well, for a final volume of 9 mL of medium.

b. Aspirate the coating solution from the Matrigel-coated plate and rinse it once with DMEM/F12.

c. Plate 1.5 mL of cells in each well of the Matrigel-coated 6-well plate.

27. Leave cells undisturbed in the incubator until the next day.

Figure 2 Generation of neural crest (NC)-derived brain pericytes from hPSCs

(A) Schematic representation of the protocol for the differentiation of hPSCs into NC-derived brain pericytes.

(B) Bright field images showing the progressive changes in cell morphology and the generation of “bridge-like” (indicated by white arrows) structures during the NC differentiation. Scale bar: 250 μm.

(C) Bright field images of the final part of the differentiation of NC into pericytes after the sorting step. The pericytes progenitor tends to progressively acquire a more mature morphology during the differentiation. Scale bar: 300 μm.

Cranial NC induction

Timing: 10 days

The purpose of this step is to direct the differentiation of hPSCs into cranial NC. NC induction is performed based on previously established protocols8,9 that use an initial SMAD inhibition in combination with WNT activation followed by an increased level of WNT activation.28. The day after plating (day 0), change the medium and switch to NC 1 medium.a. Completely aspirate the medium in every well and replace it with 1.5 mL of fresh NC 1 medium.

29. On day 1, perform a full medium change using NC 1 medium.a. Leave a thin layer of medium to avoid touching the bottom of the well with your tip during the aspiration.

30. On day 2 of differentiation, change medium and switch to NC 2 medium.a. Aspirate the medium in every well and replace it with 1.5 mL of fresh NC 2 medium.i. Leave a thin layer of medium to avoid touching the bottom of the well with your tip during the aspiration.

31. From day 2 to day 10, perform daily medium changes using NC 2 medium.

Note: After a few days, cells must show a morphological change, generating “bridge-like structures” in 3D typical of NC. The presence of “bridges” is a good indicator of the differentiation efficiency (Figure 2B). Troubleshooting 4.

32. At day 10 of NC differentiation, cells should be sorted to isolate NC cells.a. The sorting step can be performed using in-house sorting protocols. In the next section, we will describe our protocol for staining cells before sorting.

Sorting of NC cells

Timing: 1.5 h

Since the next step of pericyte differentiation requires a pure population of NC cells, a fluorescence-activated cell sorting (FACS) to enrich for NC cells is needed. The procedure to prepare cells for the FACS is described here.33. Before proceeding with the differentiation protocol, NC cells must be sorted using specific markers.Note: We sort cells for NGFR positivity, but it is possible to use different combinations of markers to make the sorting strategy more stringent.

a. Prepare the staining solution by adding the anti-NGFR antibody 1:50 into the FACS medium.

b. Conjugated antibodies are strongly recommended.

34. Prepare a single-cell suspension by using Accutase.Note: The subsequent steps will be described for cells cultured in a single well of a 6-well plate. Adjust volume accordingly to scale up or down the size the culture.

a. Aspirate the medium from cells.

b. Rinse cells with DPBS.

c. Add 500 μL of Accutase.

d. Incubate cells 5 min at 37°C.Note: Cells should be dislodged without the need for pipetting up and down several times. If this is not possible, incubate for longer periods at RT. Depending on how long the Accutase has been stored at 4°C after the thawing, the incubation time can be increased up to 20 min due to its reduced activity.

e. After the incubation, use a P1000 pipette to fully dislodge cells from the wells and gently pipette up and down to generate a single cell suspension.

f. Collect the cell suspension into a 15 mL centrifuge tube and add 5 mL of FACS medium to dilute the Accutase.

g. Move a small volume of the cell suspension into another falcon tube. These cells will be used as negative unstained control during the sorting.

35. Centrifuge cells at 200 rcf for 3 min at RT (20°C–25°C).

36. Remove the supernatant and resuspend the cells in antibody solution or FACS medium in the case of the negative control.a. The negative control can be stored at 4°C until step 42.

b. For one well of a 6-well plate we recommend using 300 μL of antibody solution or FACS medium.

37. Incubate cells with the antibody solution for 45 min on ice.a. During this time, flick the tubes several times to keep the cells in suspension.

38. After incubation, add 5 mL of FACS medium to your cells to dilute the antibody.

39. Centrifuge cells at 200 rcf for 3 min at 4°C.

40. Prepare 1 mL of DAPI solution by diluting 0.5 mg/mL DAPI (1:1000) into the FACS medium.a. Add 1 μL of DAPI to 1 mL of FACS medium.

Note: Depending on the sterility offered by the FACS facility, we recommend adding antibiotics to the FACS medium to avoid any contamination.

41. Resuspend the stained pellet in 500 μL of DAPI solution.

Note: DAPI staining allows the exclusion of dead cells from the sorting.

42. Prepare a single-cell suspension for both negative control and stained cells by using a FACS tube strainer.a. Be gentle by pipetting cells through the strainer to avoid the breaking of the strainer itself.Note: Slow volume addition to one side of the strainer facilitates the passage of the cells.

b. Store cells on ice until the sorting.

43. Sort cells using a FACS sorter by selecting the wavelength of the conjugated antibody and collect them in a 15 mL tube filled with 2 mL of FACS medium.a. Take note of the number of sorted cells for the subsequent re-plating.

Note: If, upon running the sorting of the first 5 × 105 to 1 × 106 cells, the purity of the culture is close to 99%, we suggest interrupting the process and skipping the sorting of the remaining cells to avoid the risk of contamination and reduce cellular stress.

44. After the sorting, centrifuge the collected cells at 200 rcf for 3 min at RT (20°C–25°C).

45. Aspirate the supernatant and resuspend cells in Pericyte Medium (ScienCell Research Laboratories, #1201).

Differentiation into NC-derived cortical pericytes

Timing: 7+ days

This section describes how to differentiate NC cells into pericytes by culturing cells in Pericyte Medium.46. Prepare Matrigel-coated plates or acclimatize at the RT the previously prepared dishes stored at 4°C.

47. Seed NC cells (from step 44) at 2.5 × 104/cm2 onto Matrigel-coated plates and culture them in Pericyte Medium.a. Seed 5.4 × 105 cells into a Matrigel-coated 60-mm dish into 4 mL of Pericyte Medium.

48. The day after the re-plating, perform a full medium change with fresh Pericyte Medium.Note: When performing medium change, never let the well dry, and avoid touching the cells with the pipette.

a. Pericyte precursors are replated at around 50%–60% confluency, and their morphology is shown in Figure 2C.

49. Perform medium change three times per week on a Monday, Wednesday, and Friday schedule.

50. When confluent, NC-derived pericytes can be split and expanded.

51. Passaging NC-derived cortical pericytes.Note: After the first replating, pericytes can be maintained in regular TC-treated plates.

a. Completely aspirate the medium from the plate.

b. Rinse cells with DPBS.

c. Add 1 mL of 0.05% Trypsin-EDTA and incubate cells for 3–5 min at 37°C.

d. During the incubation time, label the new plates.

e. Remove cells from the incubator and detach them from the plate using a P1000 pipette.

f. Move the cells into a 15 mL centrifuge tube.

g. Rinse the plate with 4 mL of DMEM/F12 to collect all the cells. Add the 4 mL of medium to the 15 mL centrifuge tube for a total volume of 5 mL to inactivate the Trypsin.

h. Centrifuge cells at 200 rcf for 3 min at RT.

i. Aspirate the supernatant.

j. Resuspend cells in 5 mL of Pericyte Medium.

k. Count cells using an automated cell counter.i. As an alternative, count cells manually using a Burker or Neubauer chamber.

l. For replating into a 100-mm dish, take the required volume to have 1.4 × 106 cells (2.5 × 104 cells/cm2) and add Pericyte Medium to a final volume of 10 mL.

52. At day 17 of differentiation, cells should appear with a defined pericyte-like morphology and can be used to generate Pericyte Microtissues.

Note: At this point of the differentiation, pericytes can be frozen for long-term storage in freezing medium and thawed and expanded when required.

53. Pericytes can be maintained in culture by performing medium change 3 times per week on a Monday, Wednesday, and Friday schedule and splitting them regularly when confluent.Note: We recommend building a frozen stock around day 20 of differentiation. Long-term culture will induce replicative senescence.

a. During the culture, the pericytes tend to acquire a more spindled shape (Figure 2C).

54. For freezing pericytes, collect cells as described in step 51a-i.a. Resuspend cells in Freezing medium (500 μL/vial).i. From one full 100-mm dish, you can freeze up to 3–4 vials.

b. Move the vials into a coolbox and put it at −80°C.

c. After 24 h, move the vials to liquid nitrogen.

Formation of pericyte microtissues

Timing: 1 day

In this step, it is explained how to produce pericyte microtissues that can be fused to COs for generating CP assembloids. Pericyte microtissues are generated similarly to what has been previously described for cardiac microtissues.10 Pericyte microtissues are generated by plating pericytes into a low attachment V-bottom 96-well plate.55. To generate pericyte microtissues, use hPSC-derived pericytes at 17–25 days of differentiation (see Differentiation into NC-derived cortical pericytes section).

56. Detach NC-derived cortical pericytes by trypsinization.a. Aspirate the medium from the plate completely.

b. Rinse cells with DPBS.

c. Add 1 mL of 0.05% Trypsin-EDTA and incubate cells for 3–5 min at 37°C.

d. Remove cells from the incubator and detach them from the plate by pipetting with a P1000 pipette.

e. Move the cells into a 15 mL centrifuge tube.

f. Rinse the plate with 4 mL of Pericyte Medium to collect all the cells. Add the 4 mL of Pericyte Medium to the 15 mL centrifuge tube for a total volume of 5 mL to inactivate the Trypsin.

57. Centrifuge cells at 200 rcf for 3 min at RT (20°C–25°C).

58. Aspirate supernatant and resuspend cells in 5 mL of Pericyte Medium.

59. Count cells by using a cell counter.a. Take an aliquot of 20 μL of cell suspension from the previous step and mix it 1:1 with 20 μL of Trypan blue.i. Consider the use of Erythrosin B as alternative to Trypan blue.

b. Count cells using an automated cell counter. Troubleshooting 2.i. As an alternative, count cells manually using a Burker or Neubauer chamber.

c. Take the needed volume of the cell suspension to have 1 × 106 cells.i. 1 × 106 cells are sufficient for a full 96-well plate for the generation of 96 CP assembloids.

d. Dilute the cell suspension into a final volume of 10 mL of Pericyte Medium.

60. Plate 1 × 104 cells in each well of a low attachment V-Bottom 96-well plate.a. Resuspend cells at a final dilution of 1 × 104 cells/100 μL into Pericyte Medium.i. 1 × 106 cells must be resuspended in 10 mL of medium. Adjust volume accordingly if you want to use a different number of wells.

b. Move the cell suspension to a sterile reservoir.

c. Using an 8-channel P200 pipette, seed 100 μL of cell suspension in each well of a V-Bottom 96-well plate.

61. Centrifuge the plate at 200 rcf for 10 min at RT (20°C–25°C).

62. Incubate the plate overnight (14–18 h) at 37°C and 5% CO2.

63. After the overnight incubation, pericyte microtissues should look like compact spheres (Figure 3A, left panel) and can be used to generate the cortico-pericytes (CP) assembloids.

Figure 3 Generation of cortico-pericyte (CP) assembloids

(A) Bright field pictures of the pericytes microtissue (left panel), at the moment of addition of day 14 CO to the pericyte microtissue (middle panel) and the fusion of pericyte microtissue and CO to create the CP assembloid (right panel). Scale bar: 300 μm.

(B) Bright field images showing the progressive growth of CP assembloids during the differentiation. Scale bar: 500 μm.

Generation of cortico-pericyte (CP) assembloids

Timing: 1 day

This part of the protocol describes how to generate CP assembloids by using COs on day 14 of differentiation and pericyte microtissues.64. To generate CP assembloids, add one CO at day 14 of differentiation on top of one pericyte microtissue, to allow their fusion.a. Using a sterile scalpel or scissors, cut the edge of a P1000 tip.Note: The tip should be cut to allow the aspiration of the organoid without damaging it.

b. Using a P1000 pipette, aspirate the CO from the V-bottom well generated in step 14.

c. Check the presence of the CO into the tip, to ensure its collection.

d. Release one organoid into each well of the 96-well plate containing the pericyte microtissues.Note: When moving the organoids on top of the pericyte microtissue, some CO 3 medium will be moved along with the organoid. The amount of CO 3 medium moved should be minimal to not impact the formation of CP assembloids.

65. Centrifuge the plate at 200 rcf for 1 min at RT.a. Check the presence of both the pericyte microtissue and CO in the well (Figure 3A, middle panel).

66. Incubate the plate for around 24 h at 37°C and 5% CO2.

67. The next day, check the plate to assess the fusion of the CO and the pericyte microtissue into the CP assembloid (Figure 3A, right panel).

Growing CP assembloids

Timing: 45+ days

This part focuses on culturing CP assembloids on a shaking platform in CO 3 medium to allow their maturation.68. Move the CP assembloids from the 96-well plate into a 60- or 100-mm dish.Note: The size of the plate must be chosen considering the number of assembloids you want to culture in the same plate and, more in general, the number of experimental conditions you want to test.

a. Choose the number of plates sufficient to culture all the assembloids.i. 60-mm dishes can host a maximum of 8 assembloids.

ii. 100-mm dishes can host a maximum of 24 assembloids.Note: Increasing the number of assembloids in the dish can lead to their fusion.

b. Add 6 mL or 25 mL of CO 3 medium to empty 60- or 100-mm dishes, respectively.

c. Using a sterile scalpel or scissors, cut the edge of a P1000 tip to use it in the next step.Note: The tip should be cut to allow the aspiration of the organoid without damaging it.

d. Using a P1000 pipette, aspirate the CP assembloids from the V-bottom 96-well.i. Check the presence of the CP assembloids into the tip, to ensure its aspiration from the well.

e. Release the assembloids into pre-filled dishes of step b.

69. Culture the CP assembloids at 37°C and 5% CO2 on an orbital shaker and let it rotate at approximately 75 rpm.Note: 75 rpm is related to a shaker with a 15 mm orbit rotation. In case of different orbits, adjust the speed accordingly.

a. The 60-mm dishes need a faster speed of shaking compared to the 100-mm dishes to avoid the CP assembloid fusion. We recommend increasing the rotation to 85–90 rpm. Troubleshooting 3.

70. Perform medium change 3 times per week on a Monday, Wednesday, and Friday schedule.a. After tilting the plate on one side, use a 25 mL serological pipette to carefully aspirate 20 mL (for 100-mm dishes) or 5 mL of medium (for 60-mm dishes), while avoiding aspirating the assembloids.

b. Add 20 mL (for 100-mm dishes) or 5 mL (for 60-mm dishes) of fresh CO 3 medium.

c. Position the plate back on the shaker into the incubator.

71. Monitor the assembloids growth during the maturation to ensure their correct patterning and the appearance of neuronal rosettes (Figure 3B). Troubleshooting 4.

Immunofluorescence (IF) analyses of COs and CP assembloids

Timing: 1 day

Here it is explained how to perform immunofluorescence (IF) analyses of the COs and CP assembloids.Note: In the next sections, when we refer to organoids, we intend both COs and CP assembloids.

72. IF analyses can be performed at the desired day of differentiation.

Note: To validate the generation of oRG and the expansion of the oSVZ, we suggest using organoids around day 60 of differentiation.

73. Collect the number of organoids that you wish to characterize and transfer them into a well of a 6-well plate.a. Avoid adding more than 8 organoids/well.

b. Organoids can be collected using a 50 mL serological pipette.CRITICAL: If the dimension of the organoids is too big, in order to avoid their breakage, use a sterile scalpel or scissors to cut the edge of a P1000 tip and use a P1000 pipette to aspirate the organoids.

c. You can put different conditions in different wells of the same plate.

74. Fix organoids in 4% PFA.a. Aspirate the excess medium from the well containing the organoids.

b. Rinse the organoids with 2 mL of DPBS.

c. Completely aspirate the DPBS from the wells, leaving the organoids untouched.

d. Add 1.5 mL of 4% PFA to each well.

e. Incubate the plate on shaking for 1.5 h at 37°C.i. The time can be increased to 2 h for very large organoids (>0.5 cm of diameter).

Note: The fixation at 37°C, especially for large organoids, is preferred since it provides samples of better quality for the subsequent cryo-sectioning. Do not exceed 2 hours to avoid over-fixation.

75. Remove the fixative solution and wash the organoids.a. After the fixation, aspirate the PFA and wash the organoids three times with DPBS.

Note: Remove the 4% PFA solution in a chemical hood and discard the solution in the properly assigned waste container.

76. Collect the organoids using the same procedure as step 73 and move them into a 15 mL centrifuge tube, carefully aspirating all the DPBS excess.

77. Cryoprotect organoids in sucrose solution.a. Add 4 mL of 30% sucrose solution, always freshly prepared.

b. Leave the tube containing organoids in a vertical position overnight (14–18 h) at 4°C, to allow the organoids to sink to the bottom of the tube.

78. Prepare OCT blocks of organoids.a. When sinking at the bottom, organoids can be moved from the tube into a freezing mold.

b. Use a P1000 pipette with a tip whose edge is cut off, to aspirate the organoids and move them into the freezing mold.

c. Aspirate 2 to 3 organoids together and release them in the upper part of the mold.Note: We suggest adding 1 to 3 organoids in the same mold (depending on their dimension and on the number of organoids you have).

d. Using a P1000 tip, move the organoids to the upper part of the mold and aspirate all the remaining sucrose solution.

e. Add enough OCT embedding matrix to cover the organoids and fill the mold.

f. Leave the mold for > 1 h at −80°C.Pause point: Remove the OCT samples from the mold, move them into a labeled 15 mL tube to keep them properly organized, and store them at −80°C until cryosectioning. The recommended mold can be reused indefinitely.

79. Prepare organoids cryosection and slides for immunostainings.a. Using a cryostat microtome, cut the OCT samples into 20 μm-thick sections.

b. Mount the sections on SuperFrost Plus slides.

c. Multiple consecutive sections should be mounted on serial slides to represent the various areas of the organoids on each individual slide (up to 5–8 slides for day 60 organoids).Note: The addition of consecutive slices on serial slides allows to cover the full depth of the organoid in every single slide.

d. After cutting, dry the cryosections at least for 30 min at RT (20°C–25°C).Pause point: The slides with the cryosections can be stained directly or preserved at −80°C for long-term storage.

80. Perform the staining of organoid sections on slides.a. Thaw the slides stored at −80°C for 15 min at RT (20°C–25°C).

b. Delineate the slide area with the sections with a water-repellent pen.

c. Add the slides into a closed slide holder.

d. Fill the slide holder with DPBS with Ca2+ and Mg2+ and wash the slides 3 to 5 times for 3 min at RT.Note: The washing step allows to remove the mounting leftovers that can lead to autofluorescence.

e. Dry the slides from the excess DPBS using paper tissues.Note: Avoid touching the slices with the paper tissues to avoid damaging them.

f. Add 200–300 μL of blocking solution.i. The amount of blocking solution should be enough to cover the entire area delineated with the pen in step b.

g. Incubate the slides for 1 h at RT on a flat surface to avoid the slip of the solution from the slide.

h. Prepare the primary antibody solution by diluting the primary antibodies in blocking solution.

i. Discard the blocking buffer from the slides and add 200–300 μL of primary antibody solution.

j. Incubate the slides with primary antibodies overnight (14–18 h) at 4°C on a flat surface.Note: Incubation time can be reduced to 1.5 hours at RT for some antibodies.

k. Add the slides into a closed slide holder and wash them in PBS-T washing buffer 3 times for 3 min at RT.

l. Prepare the secondary antibody solution by diluting the secondary antibodies in the diluent solution.

m. Incubate the slides with 200–300 μL of secondary antibody solution for 1 h at RT in the dark on a flat surface.

n. Add the slides into a closed slide holder and wash them in PBS-T washing buffer 3 times for at least 3 min at RT.

o. Incubate the slides in DAPI solution for 3–5 min on a flat surface.

p. Discard the DAPI solution, add the slides into a closed slide holder and wash them 3 times 3 min in PBS-T washing buffer.

q. Perform an additional wash in DPBS.

r. Using a paper tissue dry the slides carefully, avoiding touching the sections.

s. Add 1–3 drops of Prolong Mounting Medium on the slide.

t. Add a coverslip and let the slide dry from a couple of hours to overnight (14–18 h) before analyzing it.CRITICAL: To avoid the formation of bubbles, add the coverslip from one side and gently press it down.

81. When the coverslip is fully adherent, analyze the slide under a confocal microscope.

82. To validate the successful development of organoids, assess the presence and the structure of neuronal rosettes. Use SOX2 to stain neural progenitor cells, EOMES for intermediate progenitors, and TBR1 for deep-layer neurons (Figures 4A and 4B). To confirm the enrichment for oRG cells, analyze the presence of cells positive for markers such as SOX2, HOPX, GFAP, and pVIM (Figure 4C).

Figure 4 Immunofluorescence (IF) analyses of organoids

(A) Immunofluorescence images of an entire CP assembloid at day 60. The assembloids is stained for SOX2 (neural stem cells, gray), EOMES (intermediate progenitors [IPs], yellow) and TBR1 (deep-layer neurons, red). The single channels are shown. Scale bar: 500 μm.

(B) Comparison by immunofluorescence staining of the neuronal rosette structure of organoids with and without LIF treatment and CP assembloids. The treatment with LIF or the incorporation of pericytes determines the increase of the oSVZ area (SOX2 positive cells outside the ventricular zone and the layer of EOMES positive cells). The neuronal rosettes are marked with SOX2 (in gray) and EOMES (in yellow), and deep-layer neurons (TBR1 positive) are marked in red. The single channels are shown. Scale bar: 200 μm.

(C) Immunofluorescence analysis of the oRG in the neuronal rosettes. oRG cells are positive for SOX2 (gray), pVIM (green), GFAP (cyan) and HOPX (red) and are found in the oSVZ (SOX2 positive area surrounding the VZ). The single channels are shown. Scale bar: 100 μm. The image of the rosette shown here is a different perspective and magnification of the same rosette shown in the Figure 1F from Walsh et al.1

CO and CP assembloid dissociation

Timing: 3 h

In this final section, it is described how to dissociate organoids into single cells for downstream applications.Note: In the next sections, when we refer to organoids, we intend both COs and CP assembloids.

83. The protocol described here is performed using the Papain Dissociation Kit with some adaptation to the procedure indicated by the manufacturer (https://www.worthington-biochem.com/products/papain-dissociation-system/manual).

Note: The volumes are indicated for dissociating 3–8 organoids, depending on their dimension.

84. Prepare the albumin-ovomucoid inhibitor solution by adding 32 mL of EBSS (vial 1) to the albumin-ovocomucoid inhibitor mixture (vial 4). Leave at RT (20°C–25°C) until the mixture is dissolved while preparing the other components.

Note: This passage must be done only at the first use of the kit. The solution can be stored at 4°C for future dissociations.

85. Using a P1000 pipette add 500 μL of EBSS to a DNAse vial (vial 3) and pipette gently up and down for a couple of times.

CRITICAL: DNase is sensitive to shear denaturation; abstain from vigorous pipetting.

Note: The unused DNAse solution can be stored for six months at 4°C for future dissociations.

86. Add 5 mL of EBSS (vial 1) to a papain vial (vial 2). Place vial 2 in a 37°C incubator for 15 min, until papain is completely dissolved. When dissolved, the solution’s color should be brilliant pink and clear. During the incubation time, proceed with the next steps.

87. Move up to 8 organoids (depending on the organoid dimension) from the culturing plate into a clean 60-mm dish and remove the excess medium.a. Using a 50 mL serological pipette or a P1000 pipette with the tip cut at the edge, aspirate the organoids, and release them in a new plate.

b. Aspirate the excess medium, leaving organoids dry.

88. Mince each organoid using a sterile stainless-steel scalpel into 3–4 pieces.

89. When Papain is fully dissolved, add 250 μL of the DNase solution to it.a. The papain solution should look brilliant pink and clear.

b. The solution should be used fresh but can be held at RT during the procedure.

90. Add all the papain solution to the dish with the minced organoids and continue to mince them further into small pieces before the incubation.

91. Incubate the dish at 37°C for 45 min - 1 h in constant agitation on a shaking platform.a. Check the progression of the organoids’ dissociation during the incubation time to set up correctly the timing of incubation (depending on the organoid dimension, the time can be reduced or extended).

92. After the incubation, triturate the organoids by gently pipetting the solution with a P1000 pipette for 30–45 times, depending on the dimension of the organoids, until they are completely dissolved.a. If some undissociated tissue is present, allow it to settle to the bottom of the tube by gravity and then carefully aspirate the cell suspension, avoiding collecting the pellet of undissociated tissues.

93. Move the cell suspension into a 15 mL tube and centrifuge at 300 rcf for 5 min at RT.

94. During the centrifuge step, prepare the resuspension medium by mixing 540 μL EBSS (vial 1), 60 μL reconstituted albumin-ovomucoid inhibitor solution (vial 4) and 30 μL of DNase solution (vial 3) in a 15 mL centrifuge tube.

Note: In case of a high number of cells pelleted in the next step, consider preparing the double amount of the solution.

95. Discard the supernatant and immediately resuspend the cell pellet in the resuspension medium prepared in the previous step.a. Slowly add 1 mL of albumin-ovomucoid inhibitor solution on the top of the resuspended cells.i. To avoid a faster flow of medium on top of the pellet, let the medium flow drop by drop on the side of the centrifuge tube.

b. The albumin-inhibitor solution will mix with cells, creating two visible phases.i. The interface between the two layers of the gradient should be visible, although minimal mixing at this boundary does not affect the result.

96. Centrifuge the cell suspension at 70 rcf for 6 min at RT.a. Dissociated cells will form a pellet at the bottom of the tube, while membrane fragments will remain at the interface.

97. Discard the supernatant and resuspend cells in the desired medium for the subsequent analyses or experiments.

Expected outcomes

The protocol described here allows the generation of cortical forebrain organoids that contain a robustly expanded population of oRG, which results in the enlargement of the oSVZ, two features characteristic of primate cortical development.11,12 This effect can be obtained by adding exogenous human recombinant LIF or by incorporating NC-derived LIF-secreting cortical pericytes into the CO, generating the CP assembloids.

When starting from a pure population of stem cells, the described protocol allows the efficient generation of organoids that can be maintained in long-term cultures, granting their maturation. Moreover, we also detail the procedure to generate hPSC-derived pericytes and CP assembloids, providing all the tools needed to reproduce the experimental approach and perform a few downstream applications, such as IF and single-cell dissociation.

During early maturation, after 60 days of culture, the generated organoids and assembloids show markers of multiple brain cell types, closely mimicking human brain development. Markers such as SOX2 and EOMES identify respectively neural stem cells and intermediate progenitor cells, while TBR1 highlights deep-layer neurons. Conversely, the oRG population can be characterized by the expression of markers such as GFAP and HOPX, among others (Figure 4).

Extensive analyses of the LIF-treated COs and CP assembloids resulting from this protocol, are reported in Walsh et al.1

Overall, the protocol shows that increasing the cellular complexity of the organoid microenvironment is essential for improving the neocortical development recapitulation, pointing out the need to develop organoid models incorporating also non-neural cells to study their contribution to neocortex development.

Limitations

This protocol has been validated using hESC lines and hiPSC lines. However, we observed that not all hiPSC lines were equally efficient in differentiating into cortical organoids. Hence, we suggest testing the differentiation protocols in multiple hiPSC lines. In our hands, the differentiation efficiency was higher when we switched the hPSCs cultured in E8 flex medium to regular E8 medium. Despite this, the culturing of stem cells using different technical approaches (e.g., different media and coating conditions) was not formally tested.

Finally, the ability of CP assembloids in generating oRG is reduced compared to the addition of exogenous LIF, suggesting different possibilities to improve the result, such as increasing the number of pericytes or combining them with other LIF-secreting cell types. While we suggest adding pericytes as a source of LIF, they are not the sole LIF-secreting cells in the brain and microglia, for instance, also secrete LIF.13 The addition of other cellular components can represent an advancement of the proposed protocol, which could benefit both in terms of cellular complexity and physiology.

Troubleshooting

Problem 1

The presence of differentiated or contaminated hPSCs (related to steps 1, 8, and 21).

Potential solution

• If the proportion of differentiated cells in the well is less than 10%, mark the position with a marker pen, and scrape off the cells at the marked position with a P1000 pipette tip. If scraping a specific area of the plate is impossible, consider splitting cells using other reagents such as ReLeSR. Another option is reducing the incubation time of the EDTA solution or ReLeSR to induce the detachment of fewer cells. The hPSCs are usually the first ones to detach.

• Short term culture of hPSCs in 1 μM XAV939 for up to a week can reduce the presence of differentiated cells in the culture, the XAV939 can then be removed.

• During the process of culturing cells, regular detection of mycoplasma is required to ensure the healthy growth of cells.

• If there are bacterial, fungal, or mycoplasma contaminations, it is necessary to check the cell room environment, media, and consumables. Thaw a new vial of hPSC only once the environment (cell culture including incubators and hoods) has been cleaned.

Problem 2

The viability of cells is too low (related to steps 3 and 25).

Potential solution

• In case of low viability, add 10 μM Y-27632 to the DMEM/F12 used to dilute the Accutase.

• Ensure that starting hPSC cultures were not over-confluent (no greater than 80%, as per step 1) and colonies should not be merging/growing into each other.

Problem 3

The organoids fuse together (related to step 17).

Potential solution

Reduce the number of organoids in the same plate or adjust the shaking speed. The suggested rpm speeds are relative to 15-mm orbit shakers.Note: For 100-mm dishes we recommend 75 rpm and for 60-mm dishes 85–90 rpm.

Problem 4

COs do not form visible neural rosettes by day 32 (as seen in Figure 1D) or show a mistaken patterning.

Potential solution

This can be due to either improper CO patterning or properly patterned COs that fail to establish or maintain such rosettes.• Impure or spontaneously differentiating hPSC cultures, prior to the start of the experiment, can cause either issue. Refer to problem 1 for solutions.

• Culturing hPSCs in medium different than E8 can impact the differentiation efficiency. We suggest adapting cells to E8 medium prior to the start of the differentiation protocol.

• If patterning appears correct (positivity for FOXG1 expression), poor rosettes can often be the result of viability issues early in the differentiation. Ensure that there is not excessive debris following organoid plating at day 0 (Step 8; see image in Figure 1C), see problem 2 for solutions to this issue.

• Increasing the concentration of LDN193189 or XAV939 may further help with CO patterning issues, especially for hPSC lines that consistently produce poor cortical differentiations without displaying any of the issues discussed in problem 1 and problem 2.

Problem 5

The hPSCs do not differentiate efficiently into NC cells, and “bridge-like structures” are not observed (related to step 31).

Potential solution

• Plate cells at the correct density onto Matrigel-coated plates. We recommend 1.5 × 105 cells/cm2, but concentration can be altered depending on the proliferation rate of the hPSCs.

• Be sure that Matrigel-coated plates and supplements for the media have not been stored for too long at 4°C.

• Prepare the medium fresh and add the required growth factors just before use.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Arianna Baggiolini (arianna.baggiolini@ior.usi.ch).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Raffaele Luongo (raffaele.luongo@ior.usi.ch).

Materials availability

This study did not generate new unique reagents. The hESC and hiPSC lines used in this study will be made available upon request from the Studer laboratory at Memorial Sloan Kettering Cancer Center or the Baggiolini lab at the Institute of Oncology Research under a materials transfer agreement with the institute.

Data and code availability

This study did not generate new coding data.

Acknowledgments

We thank the Studer and Baggiolini lab members for their insightful comments and feedback on this manuscript. Figures contain images created with BioRender.

This work was partly supported by 10.13039/501100001711 SNSF grant 211664 (A.B.) and the Foundation for the Institute of Oncology Research (A.B.). This work was also partially supported through 10.13039/100000002 NIH grants 1R01 MH135403 , R01AG054720 , and R21NS116545 (L.S.); a grant from the 10.13039/100000005 Department of Defense (DOD), AL200169 - W81XWH2110140 (L.S.); and the 10.13039/100023920 Tri-Institutional Stem Cell Initiative .

Author contributions

A.B. and L.S. conceived and supervised the work. R.L. and A.B. wrote the manuscript and generated the figures. R.L. and R.M.W. performed and analyzed the experimental part corresponding to this protocol. A.V. optimized the organoid dissociation protocol. All authors provided feedback in editing the manuscript.

Declaration of interests

L.S. is a scientific cofounder and paid consultant of BlueRock Therapeutics, Inc. L.S. is a scientific cofounder of DaCapo Brainscience.
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References

1 Walsh R.M. Luongo R. Giacomelli E. Ciceri G. Rittenhouse C. Verrillo A. Galimberti M. Bocchi V.D. Wu Y. Xu N. Generation of human cerebral organoids with a structured outer subventricular zone Cell Rep. 43 2024 114031 10.1016/j.celrep.2024.114031
2 Pașca S.P. Arlotta P. Bateup H.S. Camp J.G. Cappello S. Gage F.H. Knoblich J.A. Kriegstein A.R. Lancaster M.A. Ming G.-L. A nomenclature consensus for nervous system organoids and assembloids Nature 609 2022 907 910 10.1038/s41586-022-05219-6 36171373
3 Florio M. Huttner W.B. Neural progenitors, neurogenesis and the evolution of the neocortex Development 141 2014 2182 2194 10.1242/dev.090571 24866113
4 Sun T. Hevner R.F. Growth and folding of the mammalian cerebral cortex: from molecules to malformations Nat. Rev. Neurosci. 15 2014 217 232 10.1038/nrn3707 24646670
5 Pellegrini L. Bonfio C. Chadwick J. Begum F. Skehel M. Lancaster M.A. Human CNS barrier-forming organoids with cerebrospinal fluid production Science 369 2020 eaaz5626 10.1126/science.aaz5626
6 Cederquist G.Y. Asciolla J.J. Tchieu J. Walsh R.M. Cornacchia D. Resh M.D. Studer L. Specification of positional identity in forebrain organoids Nat. Biotechnol. 37 2019 436 444 10.1038/s41587-019-0085-3 30936566
7 Li M. Zhong A. Wu Y. Sidharta M. Beaury M. Zhao X. Studer L. Zhou T. Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells Nat. Commun. 13 2022 6354 10.1038/s41467-022-34045-7 36302757
8 Tchieu J. Zimmer B. Fattahi F. Amin S. Zeltner N. Chen S. Studer L. A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages Cell Stem Cell 21 2017 399 410.e7 10.1016/j.stem.2017.08.015 28886367
9 Fan Y. Hackland J. Baggiolini A. Hung L.Y. Zhao H. Zumbo P. Oberst P. Minotti A.P. Hergenreder E. Najjar S. hPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung’s disease Cell Stem Cell 30 2023 264 282.e9 10.1016/j.stem.2023.02.003 36868194
10 Giacomelli E. Bellin M. Sala L. van Meer B.J. Tertoolen L.G.J. Orlova V.V. Mummery C.L. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells Development 144 2017 1008 1017 10.1242/dev.143438 28279973
11 Akula S.K. Exposito-Alonso D. Walsh C.A. Shaping the brain: The emergence of cortical structure and folding Dev. Cell 58 2023 2836 2849 10.1016/j.devcel.2023.11.004 38113850
12 Liu J. Liu W. Yang L. Wu Q. Zhang H. Fang A. Li L. Xu X. Sun L. Zhang J. The Primate-Specific Gene TMEM14B Marks Outer Radial Glia Cells and Promotes Cortical Expansion and Folding Cell Stem Cell 21 2017 635 649.e8 10.1016/j.stem.2017.08.013 29033352
13 Nakanishi M. Niidome T. Matsuda S. Akaike A. Kihara T. Sugimoto H. Microglia-derived interleukin-6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells Eur. J. Neurosci. 25 2007 649 658 10.1111/j.1460-9568.2007.05309.x 17328769
